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Introduction

Lipofectamine® RNAiMAX is a proprietary formulation specifically developed for the transfection of siRNA and Stealth™ RNAi duplexes into eukaryotic cells. Lipofectamine® RNAiMAX provides the following advantages:

High transfection efficiencies in many cell types to minimize background expression from untransfected cells and maximize knockdown

Reverse transfection and forward transfection protocols can be used for most cell lines tested. Cell-type specific transfection protocols are available at www.lifetechnologies.com/RNAi or through Technical Service.

Protocol - Reverse Transfection

Use this procedure to reverse transfect Stealth™ RNAi or siRNA into mammalian cells in a 24-well format (for other formats, see Scaling Up or Down Transfections). In reverse transfections, the complexes are prepared inside the wells, after which cells and medium are added. Reverse transfections are faster to perform than forward transfections, and are the method of choice for high-throughput transfection.
Optimize transfections as described in Optimizing Transfections, especially if transfecting a mammalian cell line for the first time. All amounts and volumes are given on a per well basis.

For each well to be transfected, prepare RNAi duplex-Lipofectamine® RNAiMAX complexes as follows.

Dilute 6 pmol RNAi duplex in 100 µl Opti-MEM® I Medium without serum in the well of the tissue culture plate. Mix gently.

Mix Lipofectamine® RNAiMAX gently before use, then add 1 µl Lipofectamine® RNAiMAX to each well containing the diluted RNAi molecules. Mix gently and incubate for 10-20 minutes at room temperature.

Dilute cells in complete growth medium without antibiotics so that 500 µl contains the appropriate number of cells to give 30-50% confluence 24 hours after plating. Use 20,000-50,000 cells/well for suspension cells.

To each well with RNAi duplex - Lipofectamine® RNAiMAX complexes, add 500 µl of the diluted cells. This gives a final volume of 600 µl and a final RNA concentration of 10 nM. Mix gently by rocking the plate back and forth.

Incubate the cells 24-72 hours at 37°C in a CO2 incubator until you are ready to assay for gene knockdown.

Assessing Transfection Efficiency

To qualitatively assess transfection efficiency, we recommend using a K1F11 Stealth™ Select RNAi (available through www.lifetechnologies.com/rnaiexpress; for human cells, oligo HSS105842 is a good choice). Adherent cells in which K1F11/Eg5 is knocked down exhibit a "rounded-up" phenotype after 24 hours due to mitotic arrest (Weil, D. et al. Biotechniques 2002, 33: 1244-1248); slow growing cells may take up to 72 hours. Alternatively, growth inhibition can be assayed after 48-72 hours. Note: The BLOCK-iT™ Fluorescent Oligo (Cat. No 2013) is optimized for use with Lipofectamine® 2000, and is not recommended for Lipofectamine® RNAiMAX.

Scaling Up or Down Transfections

To transfect cells in different tissue culture formats, vary the amounts of Lipofectamine® RNAiMAX, RNAi duplex, cells, and medium used in proportion to the relative surface area, as shown in the table.

Culture vessel

Rel. surf. area1

Vol. of plating medium

Dilution medium reverse transfection

Dilution medium forward transfection

RNAi (pmol)

RNAi (nM)

Lipofect-amine® RNAiMAX2

96-well

0.2

100 µl

20 µl

2 x 10 µl

0.12-6

1-50

0.1-0.3 µl

48-well

0.4

200 µl

40 µl

2 x 20 µl

0.24-12

1-50

0.2-0.6 µl

24-well

1

500 µl

100 µl

2 x 50 µl

0.6-30

1-50

0.5-1.5 µl

6-well

5

2.5 ml

500 µl

2 x 250 µl

3-150

1-50

2.5-7.5 µl

60 mm

10

5 ml

1 ml

2 x 500 µl

6-300

1-50

5-15 µl

100 mm

30

10 ml

2 ml

2 x 1 ml

12-600

1-50

15-35 µl

¹ Surface areas may vary depending on the manufacturer.
² If the volume of Lipofectamine® RNAiMAX is too small to dispense accurately, and you cannot pool dilutions, predilute Lipofectamine® RNAiMAX 10-fold in Opti-MEM® I Reduced Serum Medium, and dispense a 10-fold higher amount (should be at least 1.0 µl per well). Discard any unused diluted Lipofectamine® RNAiMAX.

Cotransfecting DNA and RNA using Lipofectamine® RNAiMAX

For cotransfections of plasmid DNA and Stealth™ RNAi or siRNA into mammalian cells, we recommend using Lipofectamine® 2000 (Catalog no. 11668-027), which is superior for plasmid transfections. If you want to use Lipofectamine® RNAiMAX for your cotransfections, perform a reverse transfection with the following modifications: